IgG is the most prevalent immunoglobulin class in human and other mammals and is utilised in various types of immunotherapies and diagnostic procedures. One critical issue in these therapies is the period of persistence of immunoglobulins in the circulation. The rate of clearance of an administered immunoglobulin directly affects the amount and frequency of dosage of the immunoglobulin. Studies of IgG catabolism in the circulation have identified the portions of the IgG constant domain that control IgG metabolism, including the rate of IgG degradation in the serum through interactions with FcRn (Fc receptor neonate). Increased binding affinity for FcRn increases the circulating (or serum) half-life of an IgG (see for example, Kim et al., Eur J Immunol., 24:2429 (1994)). Methods for obtaining physiologically active molecules whose half-lives are modified by introducing an FcRn-binding polypeptide into the molecules are described, for example in WO 97/43316, U.S. Pat. Nos. 5,869,046, 5,747,035, WO 96/32478. Methods for fusing the molecules to antibodies or FcRn-binding domain fragments thereof are described for example in WO 99/43713. However, the above documents do not disclose specific mutants in the IgG constant domain that affect half-life.
The modification of IgG molecules by amino acid substitution, addition or deletion to increase or reduce affinity for FcRn is disclosed in WO 98/23289, however this document does not list any specific mutants that exhibit either longer or shorter in vivo half-lives.
One mutant of the mouse IgG1 that has been shown to increase circulating half-life is the triple mutation Thr252Ala, Thr254Ser and Thr256Phe described for example in WO 97/34631. MedImmune (U.S. Pat. No. 7,083,784) have demonstrated that, in the context of human IgG1, modifications of one or more of amino acid residues 251-256, 285-290, and 308-314, within the CH2 domain and amino acid residues 385-389 and 428-436 within the CH3 domain may increase the affinity of the constant domains for FcRn and hence increase circulating half-life. In particular, they demonstrated that a triple mutation M252Y, S254T and T256E, designated “YTE” in the Fc of a human IgG1 isotype antibody can increase the circulating half-life of antibodies about 2-3 fold in non-human primates.
Characteristics of IgG4 Isotype Antibodies
IgG4 differs from other human IgG isotypes in that upon SDS-PAGE under non-reducing conditions, two protein species are observed, the major species being tetrameric IgG (H2L2, that is, two heavy chains and two light chains) and a second minor species being a “half immunoglobulin” containing a single heavy chain and a single light chain (HL). These findings indicate heterogeneity in disulphide bond formation between the two heavy chains in the hinge region. Furthermore, when different human IgG4s with different antigen-binding specificities are mixed together, the individual IgG4 molecules are able to dissociate into half immunoglobulins (HL) and which then re-associate to form tetrameric IgG (H2L2) that bind to two different antigens (bispecific antibodies). It is believed that the HL species is a major intermediate in the assembly of IgG4. Analysis of the hinge sequences of human IgG heavy chains suggested that the presence of serine at residue 228 (also referred to in some publications as residue 241; for the avoidance of doubt, this refers to the serine in the center of the IgG4 hinge region sequence CPSCP (SEQ ID NO:1)) of IgG4 (according to the numbering system of Kabat et al., Sequences of Proteins of Immunological Interest 4th Edition. Washington D.C. United States Department of Health and Human Services) may be the cause of the heterogeneity. When this residue in IgG4 is modified from serine to proline (the residue naturally found at that position in IgG1 and IgG2), it leads to the production of homogenous antibody with extended serum half-life (Angal S et al., Molecular Immunology vol 30, no 1:105-108 (1993); Labrijn et al, Nature Biotechnology vol 27, no 8:767-771; Schuurman J et al., Molecular Immunology 38 (2001) 1-8).
There is an ongoing need to generate antibodies for therapeutic purposes with enhanced properties, such as an enhanced circulating half-life.